Effect of dietary approaches to stop hypertension, and standard diets with and without curcumin on interleukin-1 alpha, 5-alpha reductase gene expressions, and androgenic and glycemic profile in polycystic ovary syndrome women undergoing in vitro fertilization treatment: A study protocol

Abstract Background Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases with major reproductive and metabolic complications with an impact on public health. Hyperandrogenism and chronic inflammation have been suggested as the leading cause of pathophysiology and clinical manifestations associated with PCOS. It seems that the altered expression of genes involved in the synthesis of pro-inflammatory cytokine and androgens contribute to the promotion of PCOS. Objective This trial aims to determine the effects of dietary approaches to stop hypertension (DASH) and standard diets with and without curcumin supplementation on the gene expression of interleukin -1 alpha(IL1α), 5α reductase and androgenic and glycemic profile among PCOS patients, who are candidates for in vitro fertilization. Materials and Methods 96 infertile women with PCOS, aged 18-40 yr, will participate in this randomized, placebo-controlled clinical trial. Based on treatment conditions and body mass index, the participants will be randomly divided into 4 equal groups using a randomized block design. They will receive a DASH or standard diet containing 52% carbohydrate, 18% protein, and 30% total fat, with the same prescribed sodium, plus 500 mg twice daily curcumin or placebo for 12 wk. The mRNA expression of IL-1 α , 5 α reductase, and androgenic and glycemic profiles will be measured at baseline and at the end of the study. Conclusion Concomitant administration of DASH diet and curcumin supplementation may reduce IL-1 α , 5 α reductase gene expressions, and improve glycemic and androgenic profiles.


Introduction
Polycystic ovary syndrome (PCOS) is an endocrine disorder that can lead to major reproductive and metabolic complications, including infertility and insulin resistance.
According to the 2003 Rotterdam criteria, the prevalence of PCOS in women of reproductive age is 4.4-18.6% (1). The Rotterdam consensus is the most widely accepted diagnostic criteria  (4). Biochemical hyperandrogenism is defined as a high concentration of testosterone and a high level of free testosterone and free androgen index (3). Hyperandrogenism may be caused by increased 5α-reductase activity, a key enzyme with an essential role in regulating the peripheral conversion of testosterone to the most active androgen, 5α-dihydrotestosterone (5). The findings showed that the activity of 5α-reductase increased in PCOS, and this high activity is related to insulin resistance (6). There are 2 isozymes of 5α reductase, encoded by the 5α-reductase type1 and type2 genes (7). Findings from an animal study suggest that insulin-mediated changes in 5α-reductase type1 messenger ribonucleic acid (mRNA) expression can negatively affect the follicle development and ovulation (8). In addition, mild chronic inflammation, independent of obesity, has been confirmed in PCOS patients (4). One of the most important inflammatory mediators, interleukin-1 (IL-1), is a multifunctional cytokine with 3 different cytokines, including cytokines (α, β) and an IL-1 receptor antagonist. IL-1 gene polymorphism can be associated with PCOS and affect ovulation, fertilization, and implantation (9,10). The dietary approaches to stop hypertension (DASH) diet has been suggested as an effective treatment option (12). DASH diet, as a lowglycemic diet (13), with a high concentration of antioxidants, can have beneficial effects in reducing insulin resistance (14), inflammation (15), and free androgen index (16). This diet is rich in vegetables, low-fat dairy products, fruits, whole grains, poultry, fish, and nuts, but is limited in foods high in sodium, cholesterol, saturated fat, red and processed meat, refined grains, sweets, and sugary drinks (12). In the last decade, the antioxidative effects of plant polyphenols have been considered (17). Indeed, among natural phenols, the therapeutic properties of curcumin have been further investigated (18). Curcumin (diferuloylmethane) is a lipophilic yellow pigment extracted from turmeric rhizomes (Curcuma longa L.) (19). Curcumin supplementation improves glycemic control and lipid metabolism, and reduces radical oxygen species in PCOS patients (20,21

Interventions
Participants will be given 2 capsules of curcumin while foods high in saturated fat and sugar will be limited (12). The amount of sodium prescribed for each diet will be the same (2300 mg per dayapproximately 1 teaspoon of salt). Overall, DASH and standard patterns will be designed using similar foods and recipes, but in different amounts, to achieve the specific characteristics of each diet.
Both diets will be planned by the study dietitian and reviewed by the Chief investigator.

Outcomes
The primary outcomes included gene expressions of IL-1α and 5α-reductase. The secondary outcomes consist serum levels of biochemical parameters comprising fasting blood sugar, fasting insulin, homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check index, as well as serum androgen concentrations (testosterone, 5αdihydrotestosterone) and anthropometric indices (height, weight, BMI, hip circumstance, waist circumstance, waist-hip ratio, waist-height ratio), body composition (muscle mass, fat mass, fat-free mass, and other factors such as trunk fat mass), physical activity, and dietary intake (energy and nutrient intake).

Demographic data and anthropometric measurements
A form including: full address, age, gender, income, employment, ethnicity, level of education, and medical history (dose and type of medicine and supplements) will be completed for each participant.
Participant's weight, height, body circumferences (waist, hip), BMI, and waist-hip ratio as well as body composition analysis (fat mass, muscle mass, fat free mass) will be measured on the first and last day of the intervention. Weight will be measured to the nearest 0.5 kg (possible errors) using a pre-calibrated electronic scale (Seca, Germany). Height will be measured without shoes with an accuracy of 0.1 cm using a Seca wall stadiometer. Then BMI will be determined as weight (kg) divided by the square of their height (m²). Waist (cm) will be measured as the distance between the lowest rib and the iliac crest. Additionally, the hip circumference will be measured around the largest part of the hip. The waist-hip ratio will be the ratio of waist to hip circumference. The waist-height ratio will be measured by dividing the waist circumstance by the height. Body composition will be determined using body composition analyzer (Tanita BC-418, Tokyo, Japan), at the beginning of the study and after 12 wk of the study. All the measurements will be repeated by a trained assessor and recorded as average.

Biochemical measurements
After 2 wk of run-in period and considering 12hr overnight fasting, 8 cc of blood samples will be collected in the morning from each participant, in the beginning, and at the end of the study. 5 cc of samples will be transferred to clotactivator tubes and then centrifuged for 30 min to separate the serum. Another 3 cc of blood samples will be collected in EDTA-coated tubes to seperate the plasma and buffy coat (the layer between red blood cells and plasma) and prepare buffy coat for gene expression assays. The allocated serum, plasma, and buffy coat will be stored immediately at -70°C. Before storage, the tripura isolation reagent will be added to the isolated buffy coat.
Serum levels of insulin, testosterone, and 5α-dihydrotestosterone will be evaluated using the enzyme-linked immunosorbent assay (ELIZA method), (ELISA kit, Monobind, California, United State). Fasting blood sugar will be measured by commercial kit using an enzymatic colorimetric method (Pars Azmoon, Tehran, Iran). Homeostasis model assessment of insulin resistance will be calculated, using the following formula:

Gene expression assay
According to the kit protocol (Favorgen Biotech Corp., Taiwan), the buffy coat fraction of whole blood cells will be used for total RNA extraction. RNA concentration will be analyzed using spectrophotometer (DS-11, DeNovix) at 260 nm. Purity will be considered as an A260/A280 ratio of about 2 (24). cDNA will be synthesized from high-quality mRNA using a cDNA synthesis kit (GeneAll Biotechnology Co.). Forward and reverse primers are selected after reviewing previous studies (25)(26)(27) and checked by primer designing tools (Primer Blast, OLIGO 7 primer analysis software), as shown in table I. IL-1α and 5α-reductase expression levels will be assessed by SYBR-green real-time PCR detection method using the kit protocol (Yekta Tajhiz Azma, IRAN), and the StepOne realtime PCR system (Applied Biosystems, Foster City, California, USA). GAPDH (glyceraldehyde phosphate dehydrogenase) will be chosen as the housekeeping gene.

Dietary intake
Food consumption information will be obtained through 3-day dietary records including 2 days per week and one weekend. Before the study, participants will be given adequate training on food consumption and will be provided with a guide including written examples for completing a questionnaire that simply explains how to accurately record food. The participants will be taught to use standard kitchen measuring tools to accurately determine the portion size. To reduce memory errors, they will be asked to make a detailed list of foods and beverages exactly when eating. The expert dietitian will review the completed dietary records, ingredient lists, and preparation methods, while will be blind to the study allocation. Each daily food record will be calculated by the Nutritionist IV software.

Physical activity
The metabolic equivalent of task-hours (METh)/day will be applied to assess physical activity questionnaire data (28).

Sample size
The sample size of 24 in each group was determined according to α = 0.05, the power of 80%, dropout rate of 20%, and considering a minimal clinically important difference of 0.969 and an effect size of 1, based on Heshmati et al. study (21) using the following formula:

Recruitment
Unique codes will be assigned to perform allocation concealment and prevent "selection bias". Supplements and placebos capsules will be provided in the same appearance (in terms of color and shape) and packed in similar bottles. The bottles will be labeled with different marks (A and B) and placed in envelopes with serial numbers by someone not related to the study. Participants and researchers will not be aware of the bottles content. Intervention groups will also receive diets from a nutritionist who is not involved in this study.
The participants will be assigned to 4 groups according to the method of stratified permuted block randomization based on treatment conditions and BMI (BMI: 18.5-25 and BMI ≥ 25), in equal block sizes of 4. The random allocation list will be created by Package 'blockrand' in R software, version 4.0.2.
Randomization and allocation will be concealed from participants and researchers until finishing the study steps and data analysis. An independent biostatistician will generate the allocation sequence. 2 midwifes will give consecutive numbers to the eligible participants and refer them to a nutritionist. The sealed envelope containing the treatment code will be opened by the supervisor, after analyzing the final data or in an emergency condition.
All trial healthcare providers, data analysts, and outcome assessors will be blinded to intervention assignments. Participants may only be aware of the type of diet but will not be aware of the placebo or curcumin supplement content.

Statistical methods
The values of quantitative variables will be presented as the mean ± standard deviation, and qualitative variables will be expressed as numbers and percentages. The normality of data will be examined by the Kolmogorov-Smirnov test.
The frequency among study groups will be compared using the Chi-square test. Independent sample t test and 2-way ANOVA will be applied to estimate the mean of quantitative variable changes and interactions between study groups; Bonferroni correction will be considered for multiple comparisons; and analysis of covariance (ANCOVA) will be used to compare the differences in the mean of quantitative data between the studied groups, while taking into account the influence of possible confounding variables. Before statistical analysis, all data will be checked for accuracy and completeness. All statistical analysis will be conducted using SPSS 22 (SPSS, Inc, Chicago, IL, USA). A p-value < 0.05 will be defined as a statistically significant level.
The "intention-to-treat" and "per protocol analysis" approaches will be performed for data analysis. The "intention-to-treat" analysis considers all participants in their main randomized groups, regardless of whether they follow the prescribed intervention according to the protocol or not, and also ignores non-compliance and withdrawal. Per protocol analysis, which accurately reflects what has been done, will be conducted based on actual adherence to the interventions.

Participants and public involvement
Participants or the public will not participate in the setting, execution, measuring outcome, or dissemination plans of our study. The study results will be provided to patients upon request. Research findings will be reported in scientific journals.

Ethical considerations
This study will be conducted following the Declaration of Helsinki. This trial was approved on September 2020 by the Medical Ethics Committee of Shahid Sadoughi University of Medical Sciences, Yazd, Iran, (Code: IR.SSU.SPH.REC.1399.145).
All goals, working methods, and possible side effects of the interventions will be explained to all Effect of diet and curcumin on PCOS the participants before starting and entering the study by the main researcher, and an informed consent form will be obtained from the participants.
Participants will be assured that all their information will be kept confidential. The contact information of one of the researchers will be provided to the participants for 24-hr contact in case of any complications or problems. The study will have no financial burden on the participants.

Monitoring
Data monitoring and safety program are considered for this study. Project managers (HM, AA) and a research advisor (MS, SJ, and AN) will supervise and monitor the research process to ensure protocol compliance and data quality. Any withdrawals, lost to follow-up and adverse events will be assessed and reported. An independent trial Data Monitoring Committee will review the collected data and evaluate clinical trials for scientific validity, safety, and integrity.

Study strengths and limitations
This will be the first study to investigate the effects of the DASH diet or curcumin supplementation on IL-1 gene expression in human. This study will be the first clinical trial that investigates the co-administration of the DASH diet and curcumin supplementation in patients with PCOS. The frequency of PCOS women with BMI: 18.5-25 is probably low, which could increase study time and lead to bias in results.

Funding
This study is Ph.D. thesis and is funded by Shahid Sadoughi University of Medical Sciences, Yazd, Iran (grant no. 8229). This trial is externally supported by Iran National Science foundation (INSF) fund after review and approval by the INSF Scientific Committee (under Project No. 99019137 dated July 6, 2021).